Researchers are striving to generate large libraries of DNA-binding molecules through rational design and combinatorial methods (see, e.g., Trauger, et al., Nature, 382, 559-561 (1996); Wemmer, et al., Curr. Opin. Struct. Biol., 7, 355-361 (1997); White, S., et al. Chem. Biol., 4, 569-578 (1997); White, et al. Nature, 391, 468-471 (1998); Boger, et al. J. Am. Chem. Soc., 122, 6382-6394 (2000)). Classes of such molecules, which include, for example, doxorubicin, daunorubicin and amsacrine, can be used to regulate gene expression and are being developed as anti-cancer drugs (Yang, et al. Pharma. Therap., 83, 181-215 (1999)). The development of high-throughput assays that can be used to quickly and efficiently evaluate these potential drug candidates is critical to progress in this area. Gold nanoparticle (Au NP)-based methods recently have been developed to screen for DNA duplex- and triplex-binding molecules both in solution and on the surface of a chip (Han, et al. Angew. Chem. Int. Ed., 45, 1807-1810 (2006); Han, et al. J. Am. Chem. Soc., 128, 4954-4955 (2006); Lytton-Jean, et al. Anal. Chem, 79, 6037-6041. (2007); and International Patent Publication No. WO 2007/047455).
Once a potential drug candidate is synthesized and its DNA binding capabilities confirmed, it is then vital to determine the selectivity of its binding for particular sequences of DNA. This information is crucial in targeting specific areas of the genome in certain therapeutic schemes (Woynarowski, Biochim. Biophys. Acta, 1587, 300-308 (2002) and Turner, et al. Curr. Drug Targets, 1, 1-14 (2000)). Unfortunately, most methods for evaluating the sequence selectivity of libraries of potential drug candidates are often inconvenient, especially for the purposes of large scale, high-throughput screening. Traditional biological techniques such as foot printing and affinity cleavage have thus far been the most commonly used methods (Trauger, et al. J. Am. Chem. Soc., 118, 6160-6166 (1996)). More recently, mass spectroscopy, competition dialysis, NMR, calorimetry, circular dichroism, and x-ray diffraction also have been used (Wan, et al. J. Am. Chem. Soc., 122, 300-307 (2000); Ren, et al. Biochemistry, 38, 16067-16075 (2000); Pelton, et al. Proc. Natl. Acad. Sci. USA, 86, 5723-5727 (1989); Rentzeperis, et al. Biochemistry, 34, 2937-2945 (1995); and Coll, et al. Proc. Natl. Acad. Sci. USA, 84, 8385-8389 (1987)). Only recently has a high-throughput, fluorescence-based method been developed to elucidate the sequence specificity of DNA-binding molecules (Boger, et al. J. Am. Chem. Soc., 123, 5878-5891 (2001) and Tse, et al. Acc. Chem. Res., 37, 61-69 (2004)).
Although useful, this fluorescent intercalator displacement (FID) assay has some weaknesses. First, the FID assay is an on-off system, where a relative decrease rather than an increase in signal is monitored. Second, the fluorescence signal of the reference intercalator (thiazole orange or ethidium bromide (EB)) often interferes with that of the DNA binding molecule of interest (Haugland, Handbook of Fluorescent Probes and Research Products. Molecular Probes, Eugene, Oreg. (2002)). Third, the fluorescence intensities of the intercalator/DNA complex are sometimes sensitive to the DNA sequence (Nygren, et al. Biopolymers, 46, 39-51 (1998)). Lastly, in the FID method, there is some error associated with the assumption that the reference intercalator has no selectivity for any particular DNA sequence. Although intercalators such as thiazole orange and ethidium bromide are considered non-specific binders, they in fact have slight preferences for certain sequences of DNA (Nygren, et al. Biopolymers, 46, 39-51 (1998) and Baguley, et al. Nucleic Acids Res., 5, 161-171 (1978)). Thus, a need exists for an efficient method of screening libraries of molecules for DNA selectivity.